Two-hybrid based screen to identify disruptive residues at multiple protein interfaces

ABSTRACT

The present invention is based, at least in part, on the development of a mating-based yeast two-hybrid screen that allows simultaneous screening for mutations that disrupt yeast two-hybrid interactions between a protein and multiple interacting partners. By coupling PCR mutagenesis and homologous recombination/gapped plasmid repair with a mating-based assay, the present invention allows screening for unique mutations that disrupt interaction with one partner, but not others. It also allows identification of specific mutations that may lie at protein-protein interfaces common to two or more partners, without employing multiple rounds of screening. In addition to screening against multiple interacting partners, the present invention removes the need for a two-step selection because truncations, frameshifts, or any mutations that affect folding are eliminated as disruptions that affect all protein partners. The methods of the present invention are named “Hotspot” because of its ability to identify “hotspot residues” in protein-protein interfaces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/326,788, filed Dec. 15, 2011, now U.S. Pat. No. 9,249,410, issuedFeb. 2, 2016, which claims the benefit of U.S. Provisional ApplicationNo. 61/423,210, filed Dec. 15, 2010, the content of each of theaforementioned applications is herein incorporated by reference in theirentirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant numberGM067180 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to the study of protein-proteininteractions, and particularly to yeast two-hybrid systems foridentifying amino acids involved in protein-protein interactions.

BACKGROUND OF THE INVENTION

Specific protein-protein interactions are essential to nearly everybiological process. Defining how specific interactions are made iscritical for a full understanding of the biological system governed bythe interacting proteins. Yeast two-hybrid analysis is a routine andpowerful assay for examining protein-protein interactions that has beenadapted to high-throughput screening (Fields, S. & Song, O. K., 340NATURE 245-46 (1989), Chien et al., 88 PROC. NATL. ACAD. SCI. U.S.A.9578-82 (1991)). In fact, large-scale yeast two-hybrid screening hasbeen utilized to collect much of the binary protein interaction dataavailable to date (Uetz et al., 403 NATURE 623-27 (2000), Ito et al., 98PROC. NATL. ACAD. SCI. U.S.A. 4569-74 (2001), Stelzl et al., 122 CELL957-68 (2005), Rual et al., 437 NATURE 1173-78 (2005), Walhout et al.,17 YEAST 88-94 (2000)). However, such large-scale yeast two-hybridscreens face numerous challenges, including strategies for cloningnumerous genes into yeast two-hybrid vectors for expressing DNA-bindingdomain (“bait”) and activation domain (“prey”) fusions in yeast.Conventional techniques involving individual restriction digests andligations for cloning genes into appropriate expression vectors can beimpractical and expensive when dealing with thousands of genes. Severalalternative systems for cloning yeast two-hybrid constructs based on DNAhomologous recombination reactions have been described to date (Uetz etal., 2000, Walhout et al., 2000). Even though these techniques eliminatethe need for restriction enzymes and ligases, they still requirethousands of yeast transformations to generate a library ofactivation-domain fusion constructs (Uetz et al., 2000, Walhout et al.,2000).

The development of mating-based screens has facilitated the conversionof yeast two-hybrid from a directed, small-scale assay to ahigh-throughput one (Gyuris et al., 75 CELL 791-803 (1993), Finley, R.L. & Brent, R., 91 PROC. NATL. ACAD. SCI. U.S.A. 12980-12984 (1994),Bendixen et al., 22 NUCLEIC ACIDS RES. 1778-79 (1994), Fromont-Racine etal., 17 YEAST 95-110 (2000)). Mating-based screens rely on haploid yeasthaving two mating types, MATa and MATα, which fuse to form diploids(Herskowitz et al., 57 MICROBIOL. REV. 536 (1988)). In these assays,DNA-binding and activation-tagged proteins are expressed in differenthaploid strains and are brought together through mating (Gyuris et al.,1993). Large numbers of individual protein-protein interactions can thenbe tested (Gyuris et al., 1993, Finley and Brent, 1994, Fromont-Racineet al., 1997, Walhout A. J. M. & Vidal, M., 24 METHODS 297-306 (2001)).However, establishing efficient strategies to mate large sets of baitand prey yeast strains to sample all possible combinations ofinteractions has also proven difficult (Uetz et al., 2000, Parrish etal., 17(4) CURR. OPIN. BIOTECHNOL. 387-93 (2006)).

Another limitation of the classic yeast two-hybrid system issimultaneous detection of multiple protein-protein interactions.Ternary-protein complexes in which a protein requires interaction with asecond protein in order to bind a third protein, and where a thirdprotein only binds to a composite site formed by the association of thefirst and second proteins, can be analyzed using a yeast “three-hybridsystem” (Zhang, Y. & Chan, D. C., 104 PROC. NATL. ACAD. SCI. U.S.A.18526-18530 (2007)). However, assays for simultaneous screening ofmultiple interacting partners have yet to be described, and currenttechnology requires multiple rounds of screening (Fromont-Racine et al.,1997).

Yeast two-hybrid assays have also been useful in mapping binaryprotein-protein interfaces (Lehming et al., 92 PROC. NATL. ACAD. SCI.U.S.A. 92, 10242-10246 (1995), Steffan et al., 18 MOL. CELL. BIOL.3752-61 (1998), Vidal et al., 93 PROC. NATL. ACAD. SCI. U.S.A. 93,10315-10320 (1996)). These assays often utilize PCR-based randommutagenesis followed by homologous recombination and gapped plasmidrepair to construct a library of mutated proteins to screen fordisruption of interaction with its binding partner (Lehming et al.,1995, Steffan et al., 1998). However, a common issue with this type ofapproach is the generation of uninformative mutations, such astruncations, frameshifts, or any mutations that affect the stability,processing or folding of the protein. To eliminate isolation of thesetrivial results, a “reverse yeast two-hybrid” system was developed(Vidal et al., 93 PROC. NATL. ACAD. SCI. U.S.A. 93, 10321-10326 (1996))to include a two-step selection process. The first step is a negativeselection for mutations that impair a protein-protein interaction andthe second step is a positive selection for a subset of those mutationsthat maintain expression of full-length stable proteins (Vidal et al.,93 PROC. NATL. ACAD. SCI. U.S.A. 93, 10321-10326 (1996)). While thistwo-step selection accomplishes both identification of disruptivemutations and elimination of trivial mutations, it can only be used tostudy the interactions of two proteins partners.

To date, mutagenesis-based yeast two-hybrid screens have only beenutilized to examine the interactions of binary protein-proteininteractions, and have not been extended to the analysis of multipleinteracting partners.

SUMMARY OF THE INVENTION

The present invention generally relates to the study of protein-proteininteractions, and particularly to yeast two-hybrid systems foridentifying amino acids involved in protein-protein interactions. Morespecifically, the present invention is based, at least in part, on thedevelopment of a mating-based yeast two-hybrid screen that allowssimultaneous screening for mutations that disrupt yeast two-hybridinteractions between a protein and multiple interacting partners. Bycoupling PCR mutagenesis and homologous recombination/gapped plasmidrepair with a mating-based assay, the present invention allows screeningfor unique mutations that disrupt interaction with one partner, but notothers. It also allows identification of specific mutations that may lieat protein-protein interfaces common to two or more partners, withoutemploying multiple rounds of screening. In addition to screening againstmultiple interacting partners, the present invention removes the needfor a two-step selection because truncations, frameshifts, or anymutations that affect folding are eliminated as disruptions that affectall protein partners. The methods of the present invention are named“Hotspot” because of its ability to identify “hotspot residues” inprotein-protein interfaces (Clackson, T. & Wells, J., 267 SCIENCE 383-86(1995)). In one embodiment, the Hotspot assay is applied to the proteinFis1, which is involved in mitochondrial fission, and its interactionswith 3 binding partnersitself, Dnm1 and Mdv1 and identified previouslyunknown interaction interfaces in these proteins.

Accordingly, in one aspect, the present invention provides methods andcompositions useful to study interactions between a protein and itsmultiple interacting partners. In one embodiment, a method foridentifying a protein interaction domain of a target protein using ayeast two-hybrid system comprises the steps of (a) mating a firsthaploid yeast cell expressing a mutant target protein with (i) a secondhaploid yeast cell expressing a first interacting partner of the targetprotein, wherein the mutant target protein is fused to a transcriptionfactor activating domain and the first interacting partner of the targetprotein is fused to the DNA binding domain of the same transcriptionfactor; and (ii) a third haploid yeast cell expressing a secondinteracting partner of the target protein, wherein the secondinteracting partner of the target protein is fused to the DNA bindingdomain of the same transcription factor; (b) selecting for diploid matedcells expressing both the mutant target protein and the first or secondinteracting partner; and selecting for yeast two-hybrid interactionsbetween the mutant target protein and the first interacting partner orthe second interacting partner, wherein a disruption of the yeasttwo-hybrid interaction indicates that the mutated amino acids of thetarget protein comprise a protein interaction domain. In anotherembodiment, steps (a)(i) and (a)(ii) are carried out simultaneously. Ina further embodiment, steps (b) and (c) are carried out simultaneouslyusing the replica plating technique. In yet another embodiment, steps(a)(i) and (a)(ii) are carried out simultaneously and wherein steps (b)and (c) are carried out simultaneously.

In a specific embodiment, the first haploid yeast cell is of mating typea and the second and third haploid yeast cells are of mating type α. Inanother embodiment, the mutant target protein comprises about 2 to about5 amino acids that are mutated relative to the wild type target protein.In a further embodiment, the method further comprises mating the firsthaploid yeast cell expressing a mutant target protein with a fourthhaploid yeast cell expressing a third interacting partner of the targetprotein, wherein the third interacting partner of the target protein isfused to the DNA binding domain of the same transcription factor.

The present invention also provides a method for using a yeasttwo-hybrid system to identify amino acid residues of a target proteinthat interact with interacting partners of the target protein comprisingthe steps of (a) providing a first haploid yeast cell expressing amutant target protein fused to either an activation domain or a DNAbinding domain of a transcription factor; (b) providing at least twohaploid yeast cells expressing different interacting partners of themutant target protein, wherein each interacting partner is fused toeither (i) an activation domain of a transcription factor if the mutanttarget protein of step (a) is fused to the DNA binding domain of thetranscription factor or (ii) a DNA binding domain of a transcriptionfactor if the mutant target protein of step (a) is fused to theactivation domain of the transcription factor; (c) mating the firsthaploid yeast cell separately with each of the at least two haploidyeast cells; (d) replica plating the mating reactions to select formated diploid yeast cells; and (e) replica plating the mating reactionsto select for yeast two-hybrid interactions, wherein a disruption of theyeast two-hybrid interaction indicates that the mutated amino acids ofthe target protein interact with an interacting partner of the targetprotein. In certain embodiments, the first haploid yeast cell is ofmating type a and the at least two haploid yeast cells expressingdifferent interacting partners of the mutant target protein are ofmating type α. In specific embodiments, the mutant target proteincomprises about 2 to about 5 amino acids that are mutated relative tothe wild type target protein.

In other embodiments, a method for using a yeast two-hybrid system toidentify amino acid residues of a target protein that interact withinteracting partners of the target protein comprises the steps of (a)separately mating a first haploid yeast cell expressing a mutant targetprotein with at least two haploid yeast cells expressing differentinteracting partners of the target protein, wherein the mutant targetprotein is fused to a transcription factor activating domain and theinteracting partners of the target protein are fused to the DNA bindingdomain of the same transcription factor; (b) selecting for diploid matedcells expressing both the mutant target protein and the first or secondinteracting partner, wherein the transcription factor activating domainand the DNA binding domain activate transcription of a reporter genewhen the target protein and the interacting partner fusion proteinsinteract; and (c) selecting for yeast two-hybrid interactions betweenthe mutant target protein and the first interacting partner or thesecond interacting partner, wherein a disruption of the yeast two-hybridinteraction indicates that the mutated amino acids of the target proteininteract with an interacting partner of the target protein. In specificembodiments, the first haploid yeast cell is of mating type a and the atleast two haploid yeast cells expressing different interacting partnersof the target protein are of mating type α. In particular embodiments,the mutant target protein comprises about 2 to about 5 amino acids thatare mutated relative to the wild type target protein.

In an alternative embodiment, a method for using a yeast two-hybridsystem to identify amino acid residues of a target protein that interactwith interacting partners of the target protein comprises the steps of(a) mating a first haploid yeast cell expressing a prey mutant targetprotein with (i) a second haploid yeast cell expressing a first baitinteracting partner of the target protein, wherein the mutant targetprotein is fused to a transcription factor activating domain and thefirst bait interacting partner of the target protein is fused to the DNAbinding domain of the same transcription factor; and (ii) a thirdhaploid yeast cell expressing a second bait interacting partner of thetarget protein, wherein the second bait interacting partner of thetarget protein is fused to the DNA binding domain of the sametranscription factor; (b) selecting for diploid mated cells expressingboth the prey mutant target protein and the first bait or second baitinteracting partner; and (c) selecting fix yeast two-hybrid interactionsbetween the prey mutant target protein and the first bait interactingpartner or the second bait interacting partner, wherein a disruption ofthe yeast two-hybrid interaction indicates that the mutated amino acidsof the target protein interact with an interacting partner of the targetprotein. In specific embodiments, the first haploid yeast cell is ofmating type a and the second and third haploid yeast cells are of matingtype α. In other embodiments, the prey mutant target protein comprisesabout 2 to about 5 amino acids that are mutated relative to the wildtype target protein.

In a specific embodiment, the present invention provides a method foridentifying amino acid residues of a hub protein that are involved inprotein-protein interaction comprising the steps of (a) providing afirst haploid yeast cell of mating type a that expresses a mutant hubprotein fused to either an activation domain or a DNA binding domain ofa transcription factor; (b) providing at least two haploid yeast cellsof mating type α that expresses different interacting partners of themutant hub protein, wherein each interacting partner is fused to either(i) an activation domain of a transcription factor if the mutant hubprotein of step (a) is fused to the DNA binding domain of thetranscription factor or (ii) a DNA binding domain of a transcriptionfactor if the mutant huh protein of step (a) is fused to the activationdomain of the transcription factor; (c) mating the first haploid yeastcell separately with each of the at least two haploid yeast cells; (d)replica plating the mating reactions to select for mated diploid yeastcells; and (e) replica plating the mating reactions to select fortwo-hybrid interactions, wherein a single disruption of the yeasttwo-hybrid interaction indicates that the mutated amino acids of the hubprotein are involved at a target protein interface with an interactingpartner, wherein a double disruption of the yeast two-hybrid interactionindicates that the mutated amino acids of the hub protein are involvedat a target protein interface with two interacting partners, and whereina disruption of the two-hybrid interaction in all selection reactionsindicates that the disruption is due to a mutation not relevant to adisruption of the interaction between the hub protein and theinteracting partners. In certain embodiments, the mutant target proteincomprises about 2 to about 5 amino acids that are mutated relative tothe wild type target protein.

In another aspect, the present invention provides methods andcompositions using a co-transformation approach to study a targetprotein's interaction with multiple interacting partners. Morespecifically, a method for using a two-hybrid system to identify aminoacid residues of a target protein that interact with interactingpartners of the target protein comprising the steps of (a)co-transforming a plurality of eukaryotic cells with (i) expressionvectors expressing a library of mutant target proteins and (ii)expression vectors expressing the interacting partners of the targetprotein, wherein each transformation reaction comprises a mutant targetprotein and an interacting partner; and (b) selecting for two-hybridinteractions between the mutant target protein and the interactingpartner, wherein a disruption of the two-hybrid interaction indicatesthat the mutated amino acids of the target protein interact with theinteracting partner. In a specific embodiment, the eukaryotic cell isyeast. In another embodiment, the eukaryotic cell is mammalian. Inparticular embodiments, the mutant target proteins are fused to anactivation domain of a transcription factor and the interacting partnersare fused to a DNA binding domain of the transcription factor.

In another specific embodiment, a method for using a two-hybrid systemto identify amino acid residues of a target protein that interact withinteracting partners of the target protein comprises the steps of (a)co-transforming eukaryotic cells with (i) an expression vector encodinga mutant target protein and (ii) an expression vector encoding aninteracting partner that interacts with the target protein; (b)repeating step (a) with expression vectors that encode N−1 of theremaining interacting partners that interact with the target protein,wherein N is the total number of interacting partners that interact withthe target protein; (c) repeating steps (a) and (b) with expressionvectors encoding other mutant target proteins; and (d) selecting fortwo-hybrid interactions, wherein a disruption of the two hybridinteraction in a selection reaction indicates that the mutated aminoacids of the target protein interact with the interacting partnerexpressed in the selection reaction. In a further embodiment, adisruption of the two hybrid interaction in all N selection reactionsfor a mutated target protein indicates that the disruption is due to amutation not relevant to a disruption of the interaction between themutant target protein and the interacting partner. In a more specificembodiment, a mutation not relevant to a disruption of the interactionbetween the mutant target protein and the interacting partner comprisesa frameshift mutation, a premature stop codon, or a mutation thatunfolds the mutant target protein. In another embodiment, the eukaryoticcell is yeast. In an alternative embodiment, the eukaryotic cell ismammalian.

In yet another embodiment, the present invention provides a method forusing a yeast two-hybrid system to identify amino acid residues of atarget protein that interact with interacting partners of the targetprotein comprising the steps of (a) providing a first haploid yeast cellof mating type a that expresses a mutant target protein fused to eitheran activation domain or a DNA binding domain of a transcription factor;(b) providing N haploid yeast cells of mating type α that express Ninteracting partners of the mutant target protein, wherein N equals thenumber of interacting partners of the target protein, and wherein eachinteracting partner is fused to either (i) an activation domain of atranscription factor if the mutant target protein of step (a) is fusedto the DNA binding domain of the transcription factor or (ii) a DNAbinding domain of a transcription factor if the mutant target protein ofstep (a) is fused to the activation domain of the transcription factor;(c) mating the first haploid yeast cell separately with each of the Nhaploid yeast cells; (d) replica plating the mating reactions to selectfor mated diploid yeast cells; and (e) replica plating the matingreactions to select for two-hybrid interactions, wherein a singledisruption of the yeast two-hybrid interaction indicates that themutated amino acids of the target protein are involved at a targetprotein interface with an interacting partner, wherein a doubledisruption of the yeast two-hybrid interaction indicates that themutated amino acids of the target protein are involved at a targetprotein interface with two interacting partners, and wherein adisruption of the two-hybrid interaction in all N selection reactionsindicates that the disruption is due to a mutation not relevant to adisruption of the interaction between the target protein and theinteracting partners.

Accordingly, the methods and compositions of the present invention canbe used to study proteins that interact with multiple partners. Incertain embodiments, the target protein is a hub protein. Signalling hubproteins that can be studied using the present invention include, butare not limited to, PDK1, Wnt, Beta-catenin, Src, Akt, Erk-1, MAPK, CDK,PTEN, PGCa, Rck1, Tra1, DISC1, Grb2, AP2, Clathrin, p53, and NFkB. Anyof the G-protein coupled receptors (GPCR) can be studied as well. SmallGTPas signaling (Ras-GRPase and Rho-GTpase), as well as cytokinesignaling including cytokines that are involved in the Stat pathway canbe studied using the methods and compositions of the present invention.Generally, the present invention can be used to study an “interactome.”The most interactive families that make up the interactome core includeP-loop containing nucleotide triphosphate hydrolases, immunoglobulins, Eset domains, trypsin-like serin proteases, winged helix DNA-bindingdomain, nucleic acid-binding domains, (Trans)glycosidases, cytochrome c,4Fe-4S ferredoxins, EGH/Laminin, EF-hand, NAD(P)-binding Rossmann-folddomains, FAD/NAD(p)-binding domain, eupredoxins, ribosomal protein S5domain 2-like, protein kinase-like (PK-like), 2Fe-2S ferredoxin-like,ARM repeat, galactose-binding domain-like, and the actin-like ATPasedomain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the Hotspot screen forsimultaneously identifying disruptive residues in one protein that showsyeast two-hybrid interactions with multiple protein partners. In thisapproach, the present inventors screened for disruptions of yeasttwo-hybrid interactions between a “hub protein” (Protein A) and “spokeproteins” (Proteins B, C, D, and so on). A mutant library of Protein Awas randomly generated using error-prone PCR. This library wastransformed into yeast with mating type a by homologous recombination.Individual colonies represent distinct mutant alleles of Protein A andwere then mated with Proteins B, C, and D in the bait strains of matintype α. Mating ensures proper plasmid transfer and the subsequentreplica plating (“repliplating”) allows for selection of disruptiveinteractions simultaneously. While this approach was demonstrated herefor three interacting partners, it could be extended for multipleinteractions. Note that Hotspot is designed to serve as its own internalcontrol by immediately identifying disruptions caused by trivialreasons, since frameshift mutations, premature stop codons, andmutations that unfold the protein result in triple disruptions thatallows rapid identification and elimination from subsequent screening.This technology was applied to the signaling hub protein Fis1, which isinvolved in mitochondrial fission, and its interactions with itself,Dnm1, and Mdv1.

FIG. 2 shows that disruptions in the Hotspot assay are identified bycolony loss on one of the three bait plates. Yeast strain L40 (Mat a)was transformed with mutagenized Fis1 prey, and single colonies wereinoculated into a 96-well plate. Each plate contains a positive control(wild-type Fis1 prey, red circle), and a negative control (empty prey,black circle). After overnight incubation in selective media, cells weremated by overnight incubation in rich medium with yeast strain AMR70(Mat α) transformed with (Top) Fis1 bait, (Middle) Dnm1 bait or (Bottom)Mdv1 bait. Cells were then repliplated onto media containing histidine(selection for mating) and onto media lacking histidine (selection foryeast two-hybrid interactions). Plates were photographed afterincubation at 30° C. for 3 days. One example of each type of disruptionis highlighted: a single disruption (blue circle), a double disruption(blue square), and a triple disruption (blue hexagon).

FIG. 3 is a schematic showing that application of Hotspot identitiesFis1 alleles that disrupt yeast two-hybrid interactions. (Left)Schematic of a subset of Fis1 alleles that contain mutations thatselectively disrupt interactions with Fis1 (top, blue), Dnm1 (middle,red), and Mdv1 (bottom, green). Each filled circle indicates a positionthat was mutated, interfacial residues previously inferred frombiochemical and structural data are highlighted in the amino acidsequence above each section using the same color scheme. (Right) Theselective disruptions identified by Hotspot are depicted on a surfacerepresentation of the Fis1 molecule (PDB entry 1y8m) for each bindingpartner. This figure was made in PyMol using a color gradient toindicate the frequency that an amino acid was identified in Hotspot.Surface representations incorporate all of the existing data from atotal of 100 alleles identified to date.

FIG. 4 demonstrates that individual Fis1 residues identified by Hotspotare essential for mitochondrial fission in vivo. (Top) Each mutantallele isolated by Hotspot as a Fis1-Mdv1 selective disruption contained2-5 amino acid mutations per Fis1 protein. Each allele was parsed intoits corresponding single point mutations for further analysis. (Bottom)Point mutations were individually subcloned into a galactose-inducibleFis1 plasmid and were tested for their ability to restore functionalfission in fis1Δfzo1Δ yeast. fis1Δfzo1Δ cells are viable when grown onthe non-fermentable carbon source, glycerol. If functional fission isrestored, these cells have unopposed mitochondrial fission and losetheir ability to metabolize glycerol from loss of mitochondrial DNAassociated with excess fission. fis1Δfzo1Δ were transformed withplasmids harboring wild-type Fis1, ΔN₁₆Fis1, or Fis1 single pointmutations. Cells were grown overnight in selective media, collected bycentrifugation, resuspended to a concentration of 1 OD₆₀₀/ml, andsubjected to 10-fold serial dilutions in water. Cells were plated ontoYP+Dextrose as a growth control and YP+Glycerol to select forrestoration of functional mitochondrial fission. Plates werephotographed after incubation at 30° C. for 5 days. Functional fissionis signified by no growth on YP+Glycerol plates.

FIG. 5 shows that Hotspot identifies Fis1 alleles that involve residuesnot previously implicated in Fis1 interactions. (Top) Schematicrepresentation of a subset of sequence variants identified by Hotspotwith selectively disrupted Fis1-Mdv1 interactions. A surfacerepresentation of the Fis1 molecule incorporating all of the existingdata from a total of 38 Fis1-Mdv1 selective disruptions is shown to theright. This figure was made in PyMOL using a color gradient to indicatethe frequency that a residue was identified by Hotspot. (Bottom) Eachallele was parsed into its corresponding single point mutations, whichwere tested individually for mitochondrial function in the yeastgrowth/death assay described in FIG. 4. Mutations that restoredfunctional fission are colored gray, while mutations that resulted innonfunctional fission are represented in dark green. Only a subset ofresidues from the mutant alleles is responsible for disrupting fission.These residues cluster in 2 main regions of Fis1, only one of which waspreviously known to be involved in Mdv1 binding. This is consistent withHotspot reporting on an intact Fis1-Mdv1 interaction. A surfacerepresentation of Fis1 mutants that disrupted mitochondrial fission invivo is shown to the right. Residues are colored according to disruptionstrength.

FIG. 6 illustrates that Fis1 residues identified as Mdv1 selectivedisruptions alter Fis1-Mdv1 interactions in vivo. A subset of Fis1mutations that were both identified by Hotspot as Mdv1 selectivedisruptions and resulted in nonfunctional fission were tested for theirability to co-immunoprecipitate Mdv1 in wild-type yeast cells. 9Myc-Fis1and 9Myc-Fis1 mutant proteins were immunoprecipitated from wild-typeyeast using anti-Myc agarose-conjugated beads and the degree of 3HA-Mdv1co-immunoprecipitation was analyzed by Western blot analysis (FIG. 6A)and quantified by densitometry (FIG. 6B). 3HA-Mdv1 co-IP levels in Bwere normalized to the amount of 3HA-Mdv1 present when wild-type9Myc-Fis1 was immunoprecipitated. FIG. 6C shows that the difference in3HA-Mdv1 co-immunoprecipitation was not due to differences in expressionlevels, as determined by TCA precipitation of yeast whole-cell lysate,followed by an anti-HA Western blot.

FIG. 7 shows that ΔN₁₆Fis1ΔTM interacts strongly with ΔN₁₆Fis1ΔTM. Dnm1,and Mdv1 by yeast two-hybrid. Yeast two-hybrid assays were performedusing the HIS3 reporter. Cells with the indicated bait and preyconstructs were grown overnight in selective media, pelleted bycentrifugation, and subjected to 10-fold serial dilutions in water, witha starting concentration of 1 OD₆₀₀/ml. Cells were spotted onto mediacontaining histidine (growth control) and media lacking histidine toselect for yeast two-hybrid interactions and plates were photographedafter incubation for 3 days at 30° C.

FIG. 8 demonstrates that colony loss in the yeast two-hybrid matingassay is not due to lack of mating or inefficient spotting. L40 (Mat a)transformed with non-mutagenized Fis1 prey was inoculated into each wellof a 96-well plate. After overnight incubation, cells were mated withAMR70 (Mat α) transformed with (A) empty bait (B) Fis1 bait (C) Dnm1bait or (D) Mdv1 bait. Cells were mated and spotted as above.

FIG. 9 shows that Fis1 mutant alleles that disrupt Fis1-Fis1, Fis1-Dnm1and Fis1-Mdv1 interactions in Hotspot are deficient in mitochondrialfission in vivo. Each mutant allele isolated by Hotspot as a Fis1-Mdv1,Fis1-Dnm1, or Fis1-Fis1 selective disruption was subcloned into agalactose-inducible Fis1 plasmid, and tested for its ability to restorefunctional fission to fis1Δfzo1Δ yeast. Cells were grown overnight inselective media, collected by centrifugation, resuspended to aconcentration of 1 OD₆₀₀/ml, and subjected to 10-fold serial dilutionsin water. Cells were plated onto YP+Dextrose as a growth control andYP+Glycerol to select for restoration of functional mitochondrialfission. Plates were photographed alter incubation at 30° C. for 5 days.This experiment was repeated twice with similar results.

FIG. 10 is a table summarizing the Hotspot screen with Fis1, Dnm1 andMdv1. Mutant alleles in each of the eight possible classes ofdisruptions (3 single, 3 double, 1 zero, and 1 triple) were identified.The total number of clones sequenced to date is 297 out of 3008 visuallyscreened colonies. The average number of amino acid changes per alleleis given in the last column. For triple disruptions, 50 clones weresequenced, but the number of amino acid changes was calculated only forclones that were full-length transcripts (denoted by an asterisk*).

FIG. 11 is a table summarizing the disruptions in Hotspot analysis ofFis1, Dnm1, and Mdv1. Note that only 2.6% of alleles identified derivedfrom frameshift mutations or premature stop codons, which occurred inthe last 18 nt of the Fis1 gene.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

I. Definitions

The terms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, etc.Modifications also include intra-molecular crosslinking and covalentattachment to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by genes may also be included in a polypeptide.

As used herein, the terms “interacting” or “interaction” means that twoprotein fragments, domains or complete proteins exhibit sufficientphysical affinity to each other so as to bring the two “interacting”protein domains or proteins physically close to each other. An extremecase of interaction is the formation of a chemical bond that results incontinual and stable proximity of the two domains or proteins.Interactions that are based solely on physical affinities, althoughusually more dynamic than chemically bonded interactions, can be equallyeffective in co-localizing two proteins. Examples of physical affinitiesand chemical bonds include but are not limited to, forces caused byelectrical charge differences, hydrophobicity, hydrogen bonds, Van derWaals force, ionic force, covalent linkages, and combinations thereof.The state of proximity between the interacting domains or entities maybe transient or permanent, reversible or irreversible. In any event, itis in contrast to and distinguishable from contact caused by naturalrandom movement of two entities. Typically although not necessarily, an“interaction” is exhibited by the binding between the interactingdomains or entities. Examples of interactions include specificinteractions between a hub protein and a spoke protein, antigen andantibody, ligand and receptor, enzyme and substrate, and the like. In aspecific embodiment, a mutant target protein (e.g., a hub protein) maybind with an interacting partner.

An “interaction” between two protein domains or complete proteins (e.g.,a hub protein and one or more interacting partners) can be determined bya number of methods. For example, an interaction can be determined byfunctional assays such as the two-hybrid system. Protein-proteininteractions can also be determined by various biochemical approachesbased on the affinity binding between two interacting partners. Suchbiochemical methods generally known in the art include, but are notlimited to, protein affinity chromatography, affinity blotting,immunoprecipitation, and the like. The binding constant for twointeracting proteins, which reflects the strength or quality of theinteraction, can also be determined using methods known in the art.

A “target protein” is a protein to be studied using the methods of thepresent invention. Typically, a target protein is a protein involved insignaling networks. In certain embodiments, a target protein is acentrally-located hub protein that controls the integration of multiplesignals into a single response. As described herein, one such protein isFis1, mitochondrial dynamics protein that regulates mitochondrialmorphology through interaction with at least 3 proteins. A targetprotein interacts with multiple interacting partners. In specificembodiments, a target protein interacts with at least two interactingpartners. A target protein can interact with about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10 or moreinteracting partners. The present invention contemplates studying theinteraction of a target protein with “N” interacting partners where Nequals any number greater than 2. In particular embodiments, and asdescribed herein, a library of mutant target proteins is generated usingtechniques known in the art (e.g., error prone PCR). This library ofmutant target proteins can be screened against N number of interactingpartners and amino acids of critical to protein-protein interactions canbe identified and studied further.

The terms “fusion protein,” “fusion polypeptide,” “fusion peptide,”“hybrid protein,” “hybrid polypeptide,” and “hybrid peptide” are usedherein interchangeably to mean a non-naturally occurring protein havinga specified polypeptide molecule covalently linked to one or morepolypeptide molecules that are not naturally linked to the specifiedpolypeptide. Thus, for example, a “fusion protein” may be two naturallyoccurring proteins or fragments (hereof linked together by a covalentlinkage. A “fusion protein” may also be a protein formed by covalentlylinking two artificial polypeptides together. Typically but notnecessarily, the two or more polypeptide molecules are “fused” togetherby a peptide bond, or linked indirectly via a linker moiety, forming asingle non-branched polypeptide chain. In particular embodiments, amutant target protein (e.g., a hub protein) is fused to a DNA bindingdomain of a transcription factor. In other embodiments, a mutant targetprotein (e.g., a hub protein) is fused to an activation domain of atranscription factor. In other embodiments, an interacting partner isfused to a DNA binding domain or an activation domain of a transcriptionfactor. In more specific embodiments, N number of interacting partnersare each fused to an activation domain of a transcription factor and alibrary of mutant target proteins (which interact with N number ofinteracting partners) are each fused to a DNA binding domain of thetranscription factor in a two-hybrid system. The number of interactingpartners may vary depending on the protein network being studied.Indeed, “N” could be an entire interactome of a hub protein.

The term “chimeric gene” refers to a non-naturally occurring nucleicacid having covalently linked together two or more distinct portionsthat are not naturally linked directly to each other. Each “chimericgene” encodes a fusion protein. In specific embodiments, a chimeric geneencodes a fusion protein comprising a mutant target protein fused toeither a DNA binding domain or an activation domain of a transcriptionfactor. In other embodiments, a chimeric gene encodes a fusion proteincomprising an interacting partner fused to either a DNA binding domainor an activation domain of a transcription factor.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter. In thepresent invention, expression vector include chimeric genes encodingfusion proteins described herein.

II. Yeast Two-Hybrid System

A “yeast two-hybrid assay” or “yeast two-hybrid system” are usedinterchangeably herein and refer to an assay or system for the detectionof interactions between protein pairs. In a typical two-hybrid screeningassay/system, a transcription factor is split into two separatefragments, the binding domain (BD) and the activation domain (AD), eachof which are provided on separate plasmids, and each of which is fusedto a protein of interest. The yeast two-hybrid assay system comprises(i) a “bait” vector, comprising a bait protein and the BD of thetranscription factor utilized in the system; (ii) a “prey” vector,comprising a prey protein (or a library of prey proteins to be screenedfor interaction with the bait protein) and the AD of the transcriptionfactor; (iii) a suitable reporter yeast strain containing the activationsequence for the transcription factor used in the system, which drivesthe expression of one or more reporter proteins. The bait and preyvectors are introduced into the reporter yeast strain, wherein theexpressed bait and prey proteins may interact. Alternatively, separatehaploid yeast strains each containing either a bait vector or a preyvector can be mated and the resulting diploid yeast strain expressesboth proteins. Interacting bait and prey protein pairs result in thereconstitution and activation of the transcription factor, which thenbinds to its compatible activation domain provided in the reporter yeaststrain, which in turn triggers the expression of the reporter gene,which may then be detected.

Any yeast cell can be used in the methods of the present invention. Inparticular embodiments, haploid cells of a yeast species within thegenus of Saccharomyces, particularly Saccharomyces cerevisiae, are used.Other examples of suitable yeast species include, but are not limitedto, Hansenula polymorpha, Pichia pastoris, and Schizosaccharomycespombe. Indeed, numerous yeast strains or derivative strains are known inthe art. Many of them have been developed specifically for certain yeasttwo-hybrid systems. The application and optional modification of suchstrains for purposes of the present invention should be apparent to askilled artisan apprised of the present disclosure. Methods forgenetically manipulating yeast strains using genetic crossing orrecombinant mutagenesis are well known in the art. See, e.g., Rothstein,101 METH, ENZYMOL., 202-11 (1983). Yeast strains that can be used in thepresent invention include, but are not limited to, L40, EGY48, andMaV103. Such strains are generally available in the research community,and can also be obtained by simple yeast genetic manipulation. Seegenerally. THE YEAST TWO-HYBRID SYSTEM, Bartel and Fields, eds., pages173-182, Oxford University Press, New York, N.Y., 1997; Kumar et al.,272 J. BIOL CHEM. 13548-13554 (1997); and Vidal et al., 93 PROC NATL.ACAD. SCI. USA, 10315-10320 (1996). In addition, the following yeast twohybrid kits are commercially available Proquest™ (Invitrogen Corp.,Carlsbad, Calif.); Matchmaker Gold (Clontech Laboratories, Inc.,Mountain View, Calif.); DUALhybrid (DualSystems Biotech AG,Switzerland); and HybriZAP 2.1 (Agilent Technologies, Inc., Santa Clara,Calif.).

In several embodiments, haploid yeast strains containing a vectorexpressing a target protein are mated with haploid yeast strainscontaining a vector expressing an interacting partner. The genetic basisof yeast mating control is well understood in the art. See e.g.,Herskowitz et al., in The Molecular and Cellular Biology of the YeastSaccharomyces: Gene Expression, Vol. 11, Jones et al., Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1992. Essentially,yeast have two haploid mating types, a and alpha. Haploid cells ofa-mating type mate with cells of alpha-mating type to form a/alphacells. Mating can be conducted in any known methods in the art. Forexample, yeast strains to be mated can be mixed in a liquid medium or ona solid medium (e.g., agar plate) to allow the a-mating type cells to bein contact with the alpha-mating type cells. In specific embodiments,mating is conducted in a relatively rich medium for a sufficient time,e.g., one hour to overnight. Selection pressure can be imposed on theyeast cells at the time of mating or after mating is completed.

To express the fusion proteins in yeast cells of the present invention,chimeric genes encoding the fusion proteins may be introduced into theyeast cell by any suitable methods known in the art. Preferably, thechimeric genes are carried in an expression vector. Each chimeric genecan be included in a separate expression vector, e.g., one expressionvector carries a chimeric gene that encodes a hub protein and a secondexpression vector carries a chimeric gene that encodes an interactingpartner. Alternatively, the two chimeric genes encoding the two fusionproteins can be included in the same expression vector. The chimericgenes may have a constitutive promoter to allow constitutive expressionof the chimeric genes to produce the fusion proteins. Inducible orrepressible promoters may also be used such that the expression of thefusion proteins can be easily controlled. Also, the expression vectorscarrying one or more chimeric genes can be maintained in the yeast cellas self-replicating extra-chromosomal elements or stably integrated intoa host chromosome.

The expression of recombinant proteins in yeast is a well developedarea, and the techniques useful in this respect are disclosed in detailin THE MOLECULAR BIOLOGY OF THE YEAST SACCHAROMYCES, Eds. Strathern etal., Vols. I and II, Cold Spring Harbor Press, 1982; Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, New York, Wiley, 1994; andGuthrie and Fink, GUIDE TO YEAST GENETICS AND MOLECULAR BIOLOGY, inMETHODS IN ENZYMOLOGY, Vol. 194, 1991, all of which are incorporatedherein by reference. Sudbery, 7 CURR. OPIN. BIOTECH. 517-524 (1996)reviews the success in the art in expressing recombinant proteins invarious yeast species; the entire content and references cited thereinare incorporated herein by reference. In addition, Bartel and Fields,eds., THE YEAST TWO-HYBRID SYSTEM, Oxford University Press, New York,N.Y., 1997 contains extensive discussions of recombinant expression offusion proteins in yeast in connection with various yeast two-hybridsystems, and cites numerous relevant references. These and other methodsknown in the art can all be used for purposes of the present invention.The application of such methods to the present invention should beapparent to a skilled artisan apprised of the present disclosure.

Generally, the vectors for recombinant expression in yeast include ayeast replication origin such as the 2μ origin or the ARSH4 sequence forthe replication and maintenance of the vectors in yeast cells. Incertain embodiments, the vectors also have a bacteria origin ofreplication (e.g., ColE1) and a bacteria selection marker (e.g., amp^(R)marker). Optionally, the CEN6 centromeric sequence is included tocontrol the replication of the vectors in yeast cells.

Any constitutive or inducible promoters capable of driving genetranscription in yeast cells may be employed to control the expressionof the chimeric genes. Such promoters are operably linked to the codingregion of the chimeric genes. Examples of suitable constitutivepromoters include, but are not limited to, the yeast ADH1, PGK1, TEF2,GPD1, HIS3, and CYC1 promoters. Examples of suitable inducible promotersinclude but are not limited to the yeast GAL1 (inducible by galactose),CUP1 (inducible by Cu^(++),) MEL1 (inducible by galactose), FUS1(inducible by pheromone) promoters; the AOX/MOX promoter from H.polymorpha and P. Pastoris (repressed by glucose or ethanol and inducedby methanol); chimeric promoters such as those that contain LexAoperators (inducible by LexA-containing transcription factors); and thelike, inducible promoters are preferred when the fusion proteins encodedby the chimeric genes are potentially toxic to the host cells.

As discussed above, in the various embodiments of the present invention,the reporter gene contains a promoter responsive to a transcriptionalactivator reconstituted from the DNA binding domain and transcriptionalactivation domain in the fusion proteins. Any transcriptional elementsknown in the art may be used so long as they confer on the reporter genethe ability to respond to a transcriptional activator or repressorreconstituted or released as a result of the interaction between twotest polypeptides in the fusion proteins expressed. Varioustranscription factors useful in yeast two-hybrid systems have beendescribed and/or are commercially available, including withoutlimitation GAL4, GCN4, ARD1, the human estrogen receptor, E. coli LexAand B42 proteins, herpes simplex virus VP16, NF-kB p65, and the like. Inaddition, hybrid transcriptional activators composed of a DNA bindingdomain from one transcriptional activator and an activation domain fromanother transcriptional activator are also known.

In particular embodiments, a transcriptional termination signal isoperably linked to the chimeric genes or the reporter genes in theexpression vectors. Generally, transcriptional termination signalsequences derived from, e.g., the CYC1 and ADH1 genes can be used.Termination sequences such as the polyadenylation signals derived frombovine growth hormone gene, SV40, lacZ and AcMNPV polyhedral genes mayalso be operably linked to the chimeric genes.

In addition, an epitope tag coding sequence for detection and/orpurification of the fusion proteins can also be incorporated into theexpression vectors. Examples of useful epitope tags include, but are notlimited to, influenza virus hemagglutinin (HA), Simian Virus 5 (V5),polyhistidine (6×His), c-myc, lacZ, GST, and the like. Proteins withpolyhistidine tags can be easily detected and/or purified with Niaffinity columns, while specific antibodies to many epitope tags aregenerally commercially available. In addition, nucleic acid sequencesencoding nuclear localization signals may also be included in a chimericgene if it is desirable for the fusion protein encoded by the chimericgene to be localized in cell nucleus.

Additionally, in particular embodiments, the expression vectors containone or more selecting markers for the selection and maintenance of onlythose yeast cells that harbor the chimeric genes. Any selectable markersknown in the art can be used for purposes of this invention so long asyeast cells expressing the chimeric gene(s) and/or reporter genes of thepresent invention can be positively identified or negatively selected.Examples of markers that can be positively identified are those based oncolor assays, including the lacZ gene which encodes β-galactosidase, thefirefly luciferase gene, secreted alkaline phosphatase, horseradishperoxidase, the blue fluorescent protein (BFP), and the greenfluorescent protein (GFP) gene. Other markers emitting fluorescence,chemiluminescence, UV absorption, infrared radiation, and the like canalso be used. Among the markers that can be selected are auxotrophicmarkers that include, but are not limited to, URA3, HIS3, TRP1, LUE2,LYS2, ADE2, and the like. Typically, for purposes of auxotrophicselection, the yeast host cells containing the bait vector and/or preyvector are cultured in a medium lacking a particular nutrient. Otherselectable markers are not based on auxotrophies, but rather onresistance or sensitivity to an antibiotic or other xenobiotic. Examplesinclude, but are not limited to, chloramphenicol acetyl transferase(CAT) gene, which confers resistance to chloramphenicol; CAN1 gene,which encodes an arginine permease and thereby renders cells sensitiveto canavanine; the bacterial kanamycin resistance gene (kan^(R)), whichrenders eucaryotic cells resistant to the aminoglycoside G418; and CYH2gene, which confers sensitivity to cycloheximide. In addition, the CUP1gene, which encodes metallothionein and thereby confers resistance tocopper, is also a suitable selection marker. Each of the above selectionmarkers may be used alone or in combination. One or more selectionmarkers can be included in a particular expression vector. As will beapparent, the selection markers used should complement the host strainsin which the expression vectors are expressed. In other words, when agene is used as a selection marker gene, a yeast strain lacking theselection marker gene (or having mutation in the corresponding gene)should be used as haploid host cells to make yeast haploid cells.

In specific embodiments, auxotrophic markers such as URA3, HIS3, TRP1,LEU2, LYS2, ADE2 and the like are used. Thus, for example, a haploidyeast cell of a-mating type expressing, a mutant target protein may bedefective in its URA3 gene (Ura⁻) and cannot grow in a medium lackinguracil. However, the haploid yeast cell has a functional HIS3 gene(His⁺). A haploid cell of alpha-mating type expressing an interactingpartner has a functional URA3 gene (Ura⁺) but is defective in HIS3(His⁻) and cannot grow on a His⁻ medium. Thus, on a medium lacking bothhistidine and uracil, neither haploid cell can grow. Only diploid cellsresulting from mating between the haploid yeast cells can form colonies.

In another embodiment, antibiotics resistance can be used as reportingmarkers. For example, a haploid yeast cell of a-mating type expressing amutant target protein may have a chloramphenicol acetyl transferase(CAT) gene, which confers resistance to chloramphenicol, but does notexpress the bacterial kanamycin resistance gene (kan^(R)), which isrequired for resistance to the aminoglycoside G418. In contrast, ahaploid cell of alpha-mating type expressing an interacting partner mayexpress the kanamycin resistance gene but not the CAT gene. Byco-culturing the two cells in a medium containing both chloramphenicoland G418 under conditions conducive to mating, the haploid cells willnot grow and only a diploid cell resulting from mating can propagate.

In addition, as described throughout, mutants of the target protein(e.g., a hub protein) are generated and used in the two-hybrid system toidentify which amino acids are involved in protein-protein interactions.In this respect, various mutations can be introduced into the targetprotein and the effect of the mutations on protein-protein interactionis examined by the above-discussed detection method. Various mutationsincluding amino acid substitutions, deletions and insertions can beintroduced into a protein sequence using conventional recombinant DNAtechnologies. In particular embodiments, a library of mutant targetproteins can be generated using error prone PCR. As described in theExamples, the present invention is designed such that disruptions of thetwo-hybrid interaction that result from frameshift mutations, prematurestop codons, mutations that unfold the target protein or other mutationsnot relevant to a disruption of the protein-protein interaction, areeasily identified and removed from further screening.

Although the foregoing description of two-hybrid systems is described inthe context of yeast, it is understood that the disclosure is not solimiting and that the present invention is applicable to other types oftwo-hybrid systems including mammalian and bacterial systems. Suchsystems are well known in the art and are commercially available. Forexample, mammalian two-hybrid systems and kits include, but are notlimited to, Mammalian Matchmaker™ (Catalog No. 630301) (ClonetechLaboratories, Inc., Mountain View, Calif.); Mammalian Two-Hybrid AssayKit (Catalog No. 211344) (Agilent Technologies, Inc., Santa Clara,Calif.); and CheckMate™ Mammalian Two-Hybrid System (Product No. E2440)(Promega Corp., Madison, Wis.) In addition, the present inventioncontemplates not only the use of the mating based yeast two-hybridsystem, but also co-transformation of yeast as well as mammalian andbacterial systems.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Materials and Methods

Plasmid Construction.

For pGADT7-Fis1ΔTM, pGADT7-ΔN₁₆Fis1ΔTM, pBHA-Fis1ΔTM, andpBHA-ΔN₁₆Fis1ΔTM, Fis1ΔTM or ΔN₁₆Fis1ΔTM were PCR amplified using PfuTurbo DNA polymerase (Agilent Stratagene, La Jolla, Calif.) frompET29b-Fis1 (Wells et a., 282 J. BIOL. CHEM. 33769-75 (2007)) asEcoRI/BamHI fragments and were subcloned into the pGADT7 and pBHA yeasttwo hybrid vectors (vectors gift of Dr. Craig Blackstone, NIH/NINDS).Dnm1 was amplified from pMAL-Dnm1 (Wells et al., 2007) as an NdeI/BamHIfragment to generate pGADT7-Dnm1 or as a SmaI/SalI fragment to generatepBHA-Dnm1. DNA encoding the full-length Mdv1 protein was cloned intopBHA and pGADT7 as an EcoRI/BamHI fragment. pGAL-Fis1 was generated frompGALGFP-Fis1 (gift of Dr. Marie Hardwick, Johns Hopkins School of PublicHealth), by PCR amplification, followed by cloning into XbaI and XhoIsites. For pRS416-MET25-9MycFis1, the MET25-9MycFis1 fragment wasremoved from pRS415-MET25-9MycFis1 (gift of Dr. Janet Shaw, Universityof Utah) with SacI and XhoI and subcloned into pRS416 (gift of Dr. JanetShaw). For pRS415-MET25-3HAMdv1, the 3HA cassette was first removed frompRS426-Dnm13HA (gift of Dr. Janet Shaw) as a NotI/SpeI fragment, andsubcloned into pRS415. To generate pRS415-MET25-3HAFis1, the 3HAcassette was then removed from pRS415 as a NotI/HindIII fragment andsubcloned into pRS415-MET25-9MycFis1, after the 9Myc cassette had beenremoved by digestion with NotI and HindIII. Finally, to generatepRS415-MET25-3HAMdv1, Mdv1 was removed from pRS415-MET25-GFP-Mdv1 (giftof Dr. Janet Shaw) as a BamHI/XhoI fragment and subcloned intopRS415-MET25-3HAFis1, after Fis1 had been removed by digestion withBamHI and XhoI. Fis1 single point mutations were introduced intopGAL-Fis1 and pRS416-MET25-9MycFis1 using the QuikChange method (AgilentStratagene). All constructs were verified by DNA sequencing.

Non-Mating Based Yeast Two-Hybrid Assays.

The yeast L40a strain (MATα, trp1, leu2, his3, LYS::lexA-HIS3,URA3::lexA-LacZ, gift of Dr. David Zappulla, Johns Hopkins University)was used to perform the yeast two-hybrid experiment shown in FIG. 7.Cells transformed with the indicated bait and prey constructs were grownovernight in -Leu/Trp selective media (MP Biomedieals, Solon, Ohio),collected by centrifugation, resuspended in water at a concentration of1 OD₆₀₀/ml and subjected to 10-fold serial dilutions in water. Growth oftransformants on -His/Leu/Trp selective media (MP Biomedicals),supplemented with 5-25 mM 3-amino-1,2,4-triazole (Sigma-Aldrich, St.Louis, Mo.) was used to measure interaction strength. Plates werephotographed after incubation at 30° C. for 3 days. At least threeindependent experiments were performed, with similar results.

PCR Mutagenesis of ΔN₁₆Fis1ΔTM.

Error-prone PCR mutagenesis was performed according to the method ofMuhlrad et al., 8 YEAST 79-82 (1992). Briefly, mutagenized ΔN₁₆Fis1ΔTMmolecules with ends homologous to pGADT7 were generated using forwardand reverse primers that each contained 36-nucleotide regions ofhomology with the pGADT7 multi-cloning site. Mutagenic PCR was performedusing Taq DNA polymerase (Invitrogen, Carlsbad, Calif.) with 0.2 μg offorward and reverse primers and 100 ng template pGADT7-ΔN₁₆Fis1ΔTM inmutagenic buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 3.5 mM MgCl₂) inwhich 0.25 mM MnCl₂ was used and dATP was limited (200 μM dCTP, 200dGTP, 200 μM dTTP, 20 μM dATP). PCR reaction conditions were: 95° C. for5:00, followed by 30 cycles of 95° C. for 1:00, 59° C. for 0:45, 70° C.for 1:30, followed by a 10 minute extension hold at 72° C. The productof the PCR reaction corresponding to the molecular weight of ΔN₁₆Fis1ΔTMwas then gel purified and used for homologous recombination.

Library Construction by Homologous Recombination.

For homologous recombination, 100 ng of gel-purified pGADT7 that hadbeen digested with EcoRI and XhoI (New England Biolabs, Ipswich, Mass.)was combined with 500 ng of gel-purified mutagenized ΔN₁₆Fis1ΔTM. Thismixture was transformed into L40a yeast, and transformants were selectedby growth on -Leu selective media. After 3 days, 3100 individualcolonies were restruck onto fresh -Leu plates and allowed to incubateovernight at 30° C. Plates were stored at 4° C. until use.

Mating-Based Yeast Two-Hybrid Assay.

Prior to performing the mating-based assay, yeast strain AMR70α (MAαT,trp1, leu2, his3, URA3::lexA-LacZ, gift of Dr. David Zappulla) wastransformed with either empty pBHA, pBHA-ΔN₁₆Fis1ΔTM, pBH-Dnm1, orpBHA-Mdv1. Transformants were selected by growth on -Trp selectivemedia. To perform the mating-based yeast two-hybrid assay, thesetransformed AMR70α (“bait”) strains were grown overnight at 30° C. in 15mL of -Trp selective media. At the same time, individual colonies ofL40a transformed with mutagenized pGADT7-ΔN₁₆Fis1ΔTM (“prey”) wereinoculated into a 96-well plate containing 225 μL -Leu selective mediaand incubated at 30° C. with shaking at 200 rpm. Each plate containedone well with a positive control (pGADT7-ΔN₁₆Fis1ΔTM) and one well witha negative control (empty pGADT7).

After overnight incubation, 50 μL of prey cells were transferred into 4new 96-well plates, 100 μL AMR70α bait strains in YPD medium at aconcentration of 1 OD₆₀₀/mL were added to each 96-well plate. Cells wereallowed to mate for 12 hours at 30° C. without shaking, and were thenrepliplated using a 10 μL slot-pin replica tool (V&P Scientific, SanDiego, Calif.) onto -Leu/Trp and -Leu/Trp/His media containing 5-25 mM3-amino-1,2,4-triazole. Cells were photographed after incubation at 30°C. for 3 days. To identify Fis1 mutations that resulted in disruptions,library plasmids were rescued from L40a and were sequenced.

Yeast Growth/Death Assay for Assessing Functionality of MitochondrialFission In Vivo.

The S. cerevisiae strain JSY5663 (MAα a fis1::HIS3 fzo1::HIS3 his3Δ200leu2Δ1 lys2Δ-202 trp1≢63 ura3-52, gift of Dr. Janet Shaw) wastransformed with pGAL-Fis1, pGAL-Fis1 point mutants, and pGAL-ΔN₁₆Fis1.Cells were grown overnight in selective media (containing dextrose),collected by centrifugation, resuspended to a concentration of 1OD₆₀₀/ml and subjected to 10-fold serial dilutions in water. Cells wereplated onto YP+Dextrose as a growth control and YP+Glycerol to selectfor restoration of functional mitochondrial fission as described(Hermann et al., 143 J. CELL BIOL. 359-73 (1998), Sesaki, H. & Jensen,R. E., 147 J. CELL BIOL. 699-706 (1999)). Plates were photographed afterincubation at 30° C. for 5 days. Two independent experiments wereperformed, with similar results.

Co Immunoprecipitation (Co-IP) Assays.

Co-IP assays were performed in the wild-type yeast strain JSY5740 (MATaura3-52 leu2Δ1 his3Δ200 trp1 Δ63, gift of Dr. Janet Shaw) transformedwith pRS416-MET25-9Myc-Fis1 or point mutants and pRS415-MET25-3HA-Mdv1.Strains were grown at 30° C. in selective media to a density of 0.7-1.0OD₆₀₀/ml, washed, resuspended in medium lacking methionine, and grown at37° C. for 2 hours. A total of 50 OD₆₀₀ units of cells were collected,and mitochondria were isolated as described previously (Zinser, E. &Daum, G. 11 YEAST 493-536 (1995)). After isolation, mitochondria weresolubilized for 1 hour at 4° C. in 400 μL IP buffer (30 mM HEPES-KOH, pH7.4, 150 mM NaCl, 1% Triton X-100, 1:500 protease inhibitor cocktail[CalBioChem, La Jolla, Calif.]). After centrifugation at 12,500 g for 10minutes, 250 μg of protein was incubated with 40 μL anti-c-Mycagarose-conjugated beads (Sigma-Aldrich, St. Louis, Mo.) for 12 hours at4° C. Agarose beads were collected, washed three times in IP buffer, andincubated in 50 μL SDS-PAGE sample buffer lacking β-mercaptoethanol at65° C. for 10 minutes to release bound proteins. After addition ofβ-mercaptoethanol and boiling, 25 μg of eluted protein was analyzed bySDS-PAGE and Western blotting with mouse monocolonal anti-Myc-HRP (9E10,Invitrogen), and rat monoclonal anti-HA (3F10, Roche Applied Science,Indianapolis, Ind.).

TCA Precipitation of Whole Cell Lysate.

Ten mL of cells in log-phase were collected by centrifugation, incubatedin 240 μL of lysis buffer (10 M NaOH, 2 mM β-mercaptoethanol, 6 mg/mlPMSF) on ice for 20 minutes. After addition of 250 μL 50%tricholoracetic acid (TCA), this mixture was incubated on ice for anadditional 20 minutes. After centrifugation for 10 minutes at 4° C., thepellet was resuspended in 500 μL 90% acetone, incubated at −20° C. for30 minutes, and pelleted by centrifugation for 10 minutes at 4° C. Thepellet was then resuspended in 60 μL TCA buffer (100 mM2-(N-morpholino)ethanesulfonic acid (MES), pH 7.4, 3M urea, 1% SDS)(Lackner et al., 325 SCIENCE 874-77 (2009)) and 6.5 mg of protein wasanalyzed by SDS-PAGE and Western blotting.

Example 1: Simultaneous Screening for Disruption of Multiple YeastTwo-Hybrid Interactions

To identify residues critical in a protein with multiple interactingpartners, a mating-based yeast two-hybrid screen (Hotspot) was designedthat allows simultaneous screening for mutations that disrupt yeasttwo-hybrid interactions between a “hub protein” (Protein A, FIG. 1) and“spoke proteins” (Proteins B, C, D, FIG. 1). In Hotspot, a pool ofrandomly generated Protein A molecules is used to create a library ofyeast two-hybrid prey plasmids by transformation into it haploid yeaststrain with mating type a by homologous recombination and gap plasmidrepair (Muhlrad et al., 1992). Individual colonies containing mutantalleles of Protein A are then mated with bait strains of mating type αcontaining Proteins B, C, and D (FIG. 1). The resulting diploids arerepliplated to select for disruption of yeast two-hybrid interactionssimultaneously (FIG. 1). As proof of principle, the Hotspot assay wasapplied to the protein Fis1 involved in mitochondrial fission and itsinteractions with 3 partner proteins—itself, Dnm1 and Mdv1. WhileHotspot is demonstrated here for three interacting partners, it can beextended for multiple interactions by increasing the number of baitstrains in the mating step (FIG. 1).

Fis1 is a mitochondrial outer membrane protein that must coordinateinteractions with at least 3 proteins (itself, Dnm1 and Mdv1) to promotemitochondrial fission in yeast. Each of these Fis1-mediated interactionsis essential for mitochondrial fission (Mozdy et al., 151 J. CELL BIOL.367-80 (2000), Karren et al., 171 J. CELL BIOL. 291-301 (2005), Lees etal., 423 J. MOL. BIOL. 143-58 (2012)). Consistent with previousbiochemical and cell biological data (Mozdy et al., 2000, Wells et al.,2007, Suzuki et al., 280 J. BIOL. CHEM. 21444-21452 (2005)), robustyeast two-hybrid interactions is observed between Fis1 and its bindingpartners when the regulatory N-terminal domain (the Fis1 arm, residues1-16) of Fis1 is removed (FIG. 7). Therefore, this strong yeasttwo-hybrid interaction was leveraged to map the Fis1-Fis1, Fis1-Dnm1 andFis1-Mdv1 interaction surfaces using the Hotspot mating-based assay.

To identify Fis1 residues important for each interaction, mutant Fis1molecules were randomly generated using error-prone PCR conditionschosen to produce 2-5 amino acid mutations in ΔN₁₆Fis1ΔTM. The PCRproducts were then gel purified according to the molecular weight ofΔN₁₆Fis1ΔTM to reduce the frequency of truncated products in the Fis1mutant pool. Next, a pGADT7-ΔN₁₆Fis1ΔTM prey library was built in theyeast two-hybrid strain L40 (mat a) by homologous recombination/gappedplasmid repair of the ΔN₁₆Fis1ΔTM PCR product with pGADT7 (Mulhrad etal., 1992, Lehming et al., 1995). After transformation, each individualL40a colony represented a distinct mutant allele of pGADT7-ΔN₁₆Fis1ΔTM.However, a transformation of L40a with gapped pGADT7 vector and noΔN₁₆Fis1ΔTM PCR product determined that approximately 6% of the librarycolonies contained repaired plasmids without the ΔN₁₆Fis1ΔTM gene. Toensure that these colonies with empty pGADT7 plasmids would not conthundinteraction results. Hotspot was performed on 94 of the backgroundcolonies obtained from control transformations. After mating andrepliplating all background colonies resulted in no growth on any of the3 bait plates (data not shown). These triple disruptions are thereforeeasily identifiable in Hotspot and can be removed from subsequentscreening.

To perform mating-based yeast two-hybrid (FIG. 1), L40a colonies withmutagenized ΔN₁₆Fis1ΔTM and mated with yeast transformed with one of thehaploid bait constructs in the opposite yeast two-hybrid mating strain(AMR70 mat α). The resulting diploids were simultaneously screened forthe disruption of each yeast two-hybrid interaction by assessing colonyloss on each of the bait plates (FIG. 2). As negative and positivecontrols, each 96-well plate included was one well containing emptypGADT7 prey and one well containing non-mutagenized pGADT7-ΔN₁₆Fis1ΔTM,respectively (FIG. 2). Cells were then repliplated onto media containinghistidine to select for mating and onto media lacking histidine toselect for yeast two-hybrid interactions. When compared to controlplates, colony loss on only one bait plate signified a singledisruption, double disruptions were identified by colony loss on twobait plates, and triple disruptions were characterized by colony loss onall bait plates (FIG. 2).

In Hotspot, mating ensures proper bait-prey plasmid transfer, and thesubsequent repliplating allows for selection of disruptive interactionssimultaneously. However, colony loss on one or more plate could be dueto lack of mating or inefficient repliplating rather than loss of theyeast two-hybrid interaction. To assess this possibility, severalcontrol Hotspot assays were performed in which wild-type ΔN₁₆Fis1ΔTMprey was inoculated into each well of the 96-well plate (FIG. 8). Aftermating and repliplating, we found that all of the colonies grew evenlyand reproducibly on all bait plates (FIG. 8). Conversely, we observed nogrowth when cells were mated with a strain carrying an empty bait vector(FIG. 8). These control experiments were performed 10 times with similarresults, suggesting that colony loss in Hotspot is due to a lack of ayeast two-hybrid interaction, and not to inefficient matins orrepliplating.

Example 2: Identification of Fis1 Critical Residues Using Hotspot

Of the 3008 visually screened colonies. Hotspot identified a number ofmutant Fis1 alleles in each of the eight possible classes of disruptions(single disruptions (3), double disruptions (2), zero disruption (1),and triple disruption (1)) (FIG. 10). Zero disruptions represented thelargest class of colonies (FIG. 10, 78.7%), followed by tripledisruptions (FIG. 10, 8.9%). Hotspot is designed to serve as its owninternal control in two important ways. First, yeast colonies that maycontain more than one mutagenized prey plasmid likely are contained inthe zero disruption class, and are thus eliminated from subsequentscreening. Hotspot also immediately identities disruptions caused bytrivial reasons, since frameshift mutations, premature stop codons,mutations that unfold the protein, and empty pGADT7 plasmids all resultin triple disruptions, which can readily be identified and eliminatedfrom subsequent screening. Of the 241 sequenced, 46 clones containedframeshift mutations or premature stop codons (FIG. 11). 89.1% of theseclones were identified in the triple disruption class, while theframeshift mutations and truncations in the remaining classes alloccurred in the last 18 nucleotides of the Fis1 gene. This analysissupports the notion that Hotspot rapidly identifies disruptions arisingfor trivial reasons in the form of triple disruptions.

Fis1-Fis1, Fis1-Dnm1 and Fis1-Mdv1 single disruptions constituted 3.4%,2.9%, and 3.2% of Hotspot clones, respectively (FIG. 10). It washypothesized that mutations that selectively disrupt Fis1-mediatedinteractions would affect residues at the site of that protein-proteininteraction and residues affected would therefore be distinct from theother classes of selective disruptions. When the point mutationscomprising the single disruptions were plotted according the amino acidsequence of Fis1, residues mutated in each class cluster in distinctregions of Fis1, with little overlap (FIG. 3). Moreover, for each classof disruptions, Hotspot identified residues previously known to beimportant in Fis1-mediated interactions, as well as regions notpreviously implicated in binding to its partners. Positions of alteredresidues in mutants with selectively disrupted homodimerization lie onregions that are not involved in Mdv1 or Dnm1 interaction (FIG. 3, bluecircles). Fis1 residues that disrupt its interaction with Dnm1 clusteron regions known to be important in Dnm1 binding (Wells et al., 2007),and on regions not been previously implicated in Dnm1 binding (FIG. 3,red circles). Residues linked to Mdv1 binding lie on both surfacesthought important for binding Dnm1 and on part of Fis1 (helix 5) thatdoes not mediate homodimerization or Dnm1 interactions (FIG. 3, greencircles). These results suggest that Hotspot accurately andsimultaneously identifies protein residues critical for binding multipleinteraction partners.

Example 3: Validation of Hotspot in a Secondary Screen for MitochondrialFunction

The present approach was validated using Fis1 alleles that selectivelydisrupted interaction with Mdv1, since this interaction is bestcharacterized to date (Karren et al., 2005, Zhang and Chan, 2007). Itwas hypothesized that Fis1 alleles identified as Fis1-Mdv1 disruptionswould affect mitochondrial fission in vivo in the context of afull-length Fis1 molecule localized to the mitochondria. To test thishypothesis, Fis1 alleles were subcloned into a galactose-inducible Fis1plasmid and were tested for their ability to restore functionalmitochondrial fission in fis1Δfzo1Δ yeast. fis1Δfzo1Δ cells are viablewhen grown on the non-fermentable carbon source, glycerol (Hermann andShaw, 1998, Sesaki and Jensen, 1999). If functional fission is restored,these cells have unopposed mitochondrial fission resulting in the lossof mitochondrial DNA that encodes oxidative phosphorylation proteinsessential for respiration on glycerol (Hermann and Shaw, 1998, Sesakiand Jensen, 1999). Functional fission, which is observed for wild-typeFis1, is signified by no growth on YP+Glycerol plates, whilenonfunctional fission, such as that seen in the non-functional ΔN₁₆Fis1,is characterized by robust growth on YP+Glycerol plates (FIG. 4). Eachof the 40 sequenced Fis1 alleles that disrupted the Fis1-Mdv1interaction in Hotspot failed to restore fission to fis1Δfzo1Δ yeast(FIG. 4, FIG. 9). These results suggest that Fis1 alleles identified byHotspot are important for mitochondrial fission in vivo in the contextof a full-length Fis1 molecule tethered to the mitochondria.

Each mutant allele isolated by Hotspot as a Fis1-Mdv1 selectivedisruption contained 1-5 amino acid mutations per ΔN₁₆Fis1 protein (FIG.10). Each of these alleles was parsed into its corresponding singlepoint mutations and individually tested the ability of each pointmutation to restore mitochondrial fission to fis1Δfzo1Δ yeast. Of the 93individual mutations that comprised the Mdv1-selective disruptivealleles, 44 mutations were able to reconstitute functional mitochondrialfission, while 49 failed to restore fission (FIG. 4). These resultssuggest that a subset of Fis1 residues identified by Hotspot isimportant for mitochondrial fission in vivo. Moreover, these residuescluster in 2 main regions of Fis1, one of which is known to be involvedin Mdv1 binding (Zhang and Chan, 2007), while the other region has notpreviously been thought to be important for Mdv1 binding (FIG. 5). Theseresults are consistent with the notion that Hotspot is reporting onintact protein-protein interactions, and that Hotspot can identify novelsites of interaction between a hub protein and its effectors.

Example 4: Validation of Hotspot by Fis1-Mdv1 Co-Immunoprecipitation inYeast

The disruption of mitochondrial fission by Fis1 mutations identified inHotspot likely arises from decreased Fis1-dependent recruitment of Mdv1to the mitochondria. To test this idea, co-immunoprecipitationexperiments were used to analyze the interaction between wild-type ormutant Fis1 proteins with Mdv1 in vivo. For these experiments, of thepoint mutants that failed to restore functional mitochondrial fission tofis1 Δfzo1Δ yeast, Fis1 mutations (Q40R, Y99D) were chosen that werepredicted to disrupt Mdv1 binding based on crystal structure of Fis1complexed with a peptide derived from Mdv1 (Griffin et al., 170 J. CELLBIOL. 237-48 (2005), Zhang and Chan, 2007) in addition to mutationsidentified here not previously thought to be important for Mdv1 binding(Y45H). Co-IP assays to test the ability of Hotspot mutations with Fis1were performed on mitochondria isolated from wild-type yeast expressing3HA-Mdv1 and either 9Myc-Fis1 or 9Myc-Fis1 mutants. Proteins wereimmunoprecipitated with anti-Myc-conjugated beads, and eluted proteinswere analyzed by Western blotting with anti-HA antibodies. 9Myc-Fis1 andmutants were all effectively immunoprecipitated from solubilizedmitochondria (FIG. 6A, Lanes 1, 306). As reported previously, Mdv1co-immunoprecipitates with 9Myc-Fis1 in an antibody-dependent manner(Tieu et al., 158 J. CELL BIOL. 445-52 (2002), Karren et al., 2005, FIG.6A, Lanes 1-3). However, less Mdv1 co-immunoprecipated was observed whenHotspot mutations were introduced into 9Myc-Fis1 (FIG. 6A, Lanes 4-6).The Q4OR and Y99D HOTSPOT mutants, which corresponded to residuespreviously identified as important for Mdv1 binding, disrupted Mdv1co-immunoprecipitation by 30% and 90%, respectively (FIG. 6B). Moreover,the HOTSPOT mutant (Y45H) not previously identified as important forMdv1 binding, also disrupted Mdv1 co-immunoprecipitation to a similardegree as the Y99D mutant (FIG. 6B), suggesting that mutationsidentified by HOTSPOT are indeed important for binding Mdv1.

We claim:
 1. A method for identifying a protein interaction domain of atarget protein using a yeast two-hybrid system comprising the steps of:(a) providing a first haploid yeast cell expressing a mutant targetprotein fused to either an activation domain or a DNA binding domain ofa transcription factor; (b) providing at least two haploid yeast cellsof opposite mating type than the first haploid yeast cell of step (a)that express different interacting partners of the mutant targetprotein, wherein each interacting partner is fused to either (i) anactivation domain of the transcription factor if the mutant targetprotein of step (a) is fused to the DNA binding domain of thetranscription factor or (ii) a DNA binding domain of the transcriptionfactor if the mutant target protein of step (a) is fused to theactivation domain of the transcription factor; (c) mating the firsthaploid yeast cell of step (a) separately with each of the at least twohaploid yeast cells of step (b); (d) replica plating the matingreactions to select for mated diploid yeast cells; and (e) replicaplating the mating reactions to select for two-hybrid interactions,wherein a disruption of the yeast two-hybrid interaction in a selectionreaction indicates that the mutated amino acids of the target proteincomprise a protein interaction domain that interacts with theinteracting partner expressed in the selection reaction and wherein adisruption of the two-hybrid interaction in all selection reactions thatresults in colony loss indicates that the disruption is likely due to amutation that affects the stability, processing or folding of the targetprotein.
 2. The method of claim 1, wherein steps (d) and (e) are carriedout simultaneously.
 3. The method of claim 1, wherein the mutant targetprotein comprises about 2 to about 5 amino acids that are mutatedrelative to the wild type target protein.
 4. A method for using a yeasttwo-hybrid system to identify amino acid residues of a target proteinthat interact with interacting partners of the target protein comprisingthe steps of: (a) providing a first haploid yeast cell of mating type athat expresses a mutant target protein fused to either an activationdomain or a DNA binding domain of a transcription factor; (b) providingN haploid yeast cells of mating type a that express N interactingpartners of the mutant target protein, wherein N equals the number ofinteracting partners of the target protein, and wherein each interactingpartner is fused to either (i) an activation domain of a transcriptionfactor if the mutant target protein of step (a) is fused to the DNAbinding domain of the transcription factor or (ii) a DNA binding domainof a transcription factor if the mutant target protein of step (a) isfused to the activation domain of the transcription factor; (c) matingthe first haploid yeast cell separately with each of the N haploid yeastcells; (d) replica plating the mating reactions to select for mateddiploid yeast cells; and (e) replica plating the mating reactions toselect for two-hybrid interactions, wherein a disruption of the yeasttwo-hybrid interaction in a selection reaction indicates that themutated amino acids of the target protein interact with the interactingpartner expressed in the selection reaction and wherein a disruption ofthe two-hybrid interaction in all N selection reactions indicates thatthe disruption is due to a mutation not relevant to a disruption of theinteraction between the target protein and the interacting partners.